Ricin toxin is a naturally occurring toxin that is derived from the seeds of Ricinus communis, commonly known as castor beans. It is composed of an enzymatically active cytotoxic polypeptide chain, commonly called the "A" chain and referred to herein as "RTA", that is bound by a single disulfide link to a second polypeptide chain commonly called the "B" chain that is presumed to be responsible for binding the toxin molecule to cells and aiding in translocating RTA into the cytoplasm. RTA is capable of catalytically inactivating the large subunit of ribosomes in vitro and the mechanism of RTA for in vivo cytotoxicity is believed to reside in this capacity for ribosome inactivation.
Olsnes, S. Perspectives in Toxicology, A. W. Bernheimer, Ed (1977) J. Wiley & Sons, NY, pp 122-147 and Olsnes, S., et al, Molecular Action of Toxins and Viruses, Cohen et al., Ed (1982) Elsevier, Amsterdam, pp 51-105 characterize native RTA as having an apparent molecular weight of 32,000. Copending, commonly owned U.S. patent application Ser. No. 715,934 filed Mar. 25, 1985 and a continuation-in-part thereof, Ser. No. 837,583 filed Mar. 7, 1986, disclose the native structural gene for RTA, the deduced amino acid sequence of RTA, DNA constructs for cloning and expressing the RTA gene, and transformed bacteria capable of synthesizing intracellularly produced, soluble recombinant RTA (srRTA). Those patent applications further describe the production of such recombinant RTA by such bacteria and a procedure for recovering RTA from the bacteria.
Copending, commonly owned U.S. patent application Ser. No. 905,283, filed Sept. 9, 1986, describes a method for recovering substantially pure, soluble recombinant RTA (srRTA) in high yields from microorganisms transformed to express RTA.
Copending, commonly owned U.S. patent application Ser. No. 806,256 filed Dec. 6, 1985, and a continuation-in-part thereof, U.S. Ser. No. 913,357 filed Sept. 30, 1986, disclose immunoconjugates comprising murine monoclonal antibodies conjugated to RTA which are effective against human ovarian tumors.
Copending, commonly owned U.S. patent application Ser. No. 690,750 filed Jan. 11, 1985 and a continuation-in-part thereof, U.S. Ser. No. 842,476 filed Mar. 21, 1986, disclose immunoconjugates comprising murine monoclonal antibodies conjugated to RTA which are effective against human breast tumors.
Native RTA high purified by affinity chromatography is available from SIGMA chemical company in a 40% glycerol solution at pH 6.0 containing 10 mM phosphate, 0.15 M NaCl, 10 mM galactose and 0.5 mM dithioerythritol.
RTA subunits of the ricin toxin whether native or recombinant, when not attached to the ricin toxin B chain (RTB) subunit, are hydrophobic. In formulations containing water, the RTA subunits tend to adhere to each other and aggregate at the water/air interface and around air bubbles. The stability problems associated with RTA also affect RTA-immunoconjugate preparations. The instant invention provides solutions to such stability problems.